The CD1 system of MHC class I-like proteins is firmly established as a group of antigen presenting molecules that activates T cells specific for lipids and glycolipids. A population of T lymphocytes known as NK T cells recognizes specific lipid ligands presented by the CD1d protein, and this component of the CD1-dependent immune response is highly conserved between humans and mice. Many detailed studies in mouse models have shown that CD1d-restricted NK T cells contribute to immune responses against pathogens, the elimination of malignant tumors, and the prevention of autoimmune diseases. A synthetic alpha-galactosyl ceramide known as KRN7000 has been identified as a glycolipid ligand that binds to CD1d and strongly activates multiple effector functions of NK T cells. Through collaborative efforts with established experts in the area of synthetic organic chemistry, the applicant has developed novel approaches to the creation of alpha-galactosyl ceramides with a range of structural alterations. Many of these compounds preserve the ability to activate CD1d-restricted NK T cells, and preliminary studies indicate that the have immunomodulatory activities that are significantly different from those of KRN7000. The current proposal will investigate the immunomodulatory activities of a large panel of novel alpha-galactosyl ceramides. Properties of these compounds to be investigated include induction of Th1 (inflammatory) versus Th2 (anti-inflammatory) cytokine production, specific targeting of NK T cell activation by certain types of antigen presenting cells or in certain tissues, and their ability to cause expansion as opposed to apoptosis of NK T cells. The proposed experiments will include in vitro cell culture studies of both human and murine NK T cells, and in vivo studies using mice. There is a high expectation that the proposed studies will contribute directly to the development of clinically useful immunomodulatory agents that act on NK T cells.